Genetic and biochemical investigations using the CYC7-H2 regulatory mutation in the eukaryotic organism Saccharomyces cerevisiae are proposed to elucidate the molecular interactions of transposable elements that define their role in regulation of gene expression. The CYC7-H2 mutation was caused by insertion of the yeast transposable element, Ty1, in the control region of the CYC7 locus, which is the structural gene for iso-2-cytochrome c. The insertion causes a 20-fold overproduction of iso-2-cytochrome c that is regulated by trans-acting loci which normally control conjugation in yeast. The specific aim of the proposed studies is to map the structural regions of the Ty1 element that are responsible for this abnormal gene expression and regulation. The cloned CYC7-H2 gene will be used to obtain deletions and rearrangements of defined segments of the transposable element in vitro by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. Recombinant plasmids with the various modified CYC7-H2 genes inserted in a yeast integrating vector will be used to transform a cytochrome c deficient yeast strain. Gene expression will be compared in the resulting transformed strains by (1) quantitative determination of iso-2-cytochrome c produced and (2) qualitative characterization of steady-state iso-2-cytochrome c RNA molecules. The CYC7-H2 derivatives will also be tested for response to trans-acting regulatory loci. Functional segments of the transposable element that are identified by these methods will be the focus of future investigations. Projected studies would define molecular interactions involving these functional segments that regulate gene expression. Such mechanisms are relevant to certain models for oncogene expression involving RNA tumor viruses.